D. van der Waaij

Colonization resistance of the digestive tract in conventional and antibiotic-treated mice

SUMMARY The effect of oral administration of antibiotics on the intestinal flora of conventional mice and their resistance to colonization by orally introduced Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa was studied. Colonization resistance (CR) was expressed as the log of the oral bacterial dose followed by a persistent take in 50 % of the contaminated animals. The intestinal flora was virtually eliminated by the antibiotics and this elimination was accompanied by a precipitous fall of CR. CR gradually returned to normal values during the period of repopulation of the intestinal tract by the organisms surviving the treatment. Antibiotic treatment resulted in the disappearance of Enterobacteriaceae, enterococci, staphylococci and yeasts and, under appropriate housing conditions, the animals remained free of these organisms indefinitely. Germ-free mice contaminated with the intestinal flora of an antibiotic-treated animal and their offspring housed in a germ-free isolator showed high values of CR. Their intestinal flora consisted of anaerobic bacteria only. Apparently, these anaerobes are responsible for CR in these and in conventional mice.

In vivo IgA coating of anaerobic bacteria in human faeces.

The bacterial flora in the human colon, although extremely diverse, has a relatively stable composition and non-infectious anaerobic bacteria are dominant. The flora forms a pool of numerous different antigens separated from mucosal immunocompetent cells by just a single layer of epithelial cells. Despite this thin barrier, however, the colonic mucosa is physiologically only mildly inflamed. This study looked at the mucosal humoral immune response against faecal anaerobes. By flow cytometric analysis the in vivo immunoglobulin coating of anaerobic bacteria in faecal samples of 22 healthy human volunteers was determined. In a previous study flow cytometric analysis of faecal bacteria has been found to be a very sensitive method to detect immunoglobulins on faecal bacteria. This technique showed that in vivo many bacteria are coated with IgA (24-74%) and less with IgG and IgM. The presence of many bacteria coated with IgA implies that IgA coating does not result in permanent removal of the species from the colon. The absence of immunoglobulin coating suggests that there is immunological unresponsiveness for anaerobic bacterial antigens. It is concluded that both immunological unresponsiveness and preferential coating with IgA are responsible for the relative absence of colonic mucosal inflammation.

Protective isolation and antimicrobial decontamination in patients with high susceptibility to infection

SummaryThe efficiency of protective isolation and protective isolation plus gastrointestinal decontamination on the control of infectious complications in patients with decreased defence capacity was investigated prospectively in a cooperative trial with the participation of clinical centers in several European countries (European Organization for Research on Treatment of Cancer, Gnotobiotic Project Group). The study was performed in patients with acute leukemia under remission-induction therapy on the basis of the frequency of such patients in the participating centers. Over a period of five years, 137 cases of acute leukemia were randomly allocated to three different treatment groups as follows: Group A: strict protective isolation in plastic isolation systems or laminar air flow isolators and prophylactic antimicrobial decontamination by non-absorbable antibiotics, Group B: strict isolation alone in one or the other type of isolator, and Group C: routine hospital ward. The results demonstrated that the incidence of contamination and colonization with new bacteria could be decreased significantly by strict protective isolation alone and even more markedly by strict protective isolation and antimicrobial decontamination. They also demonstrated that in Group A there were less frequent episodes of severe infection. More specifically, the incidence of pulmonary infection was reduced significantly in both Groups A and B in comparison with Group C. The remission rate of acute leukemia was higher in patients of Groups A and B versus Group C (69%, 61%, 49%), although this result was not significant. However, the microbiological investigations and the outcome of the study suggest that the techniques of protective isolation and antimicrobial decontamination have to be improved to decrease further the incidence of infection in the compromised host.ZusammenfassungDie Wirksamkeit der protektiven Isolierung und Isolierung mit zusätzlicher gastrointestinaler Dekontamination zur Beherrschung von infektiösen Komplikationen bei Patienten mit verminderter Abwehrfähigkeit wurde prospektiv in einer Gemeinschaftsstudie unter Beteiligung von einigen klinischen Zentren in einigen europäischen Ländern durchgeführt (European Organisation for Research on Treatment of Cancer, Gnotobiotic Project Group). Die Untersuchung wurde bei Patienten mit akuter Leukämie durchgeführt bei einer zur Remission führenden Therapie. Die Patientenzahl hing von dem in den verschiedenen Zentren zur Verfügung stehenden Krankengut ab. Über einen Zeitraum von fünf Jahren wurden 137 Fälle von drei verschiedenen Behandlungsgruppen randomisiert zugeteilt: Gruppe A (strenge protektive Isolierungssysteme mit Plastikzelten oder Laminar-Airflow-Isoliereinheiten und prophylaktische antimikrobielle Dekontamination durch Antibiotika, die nicht absorbiert werden), Gruppe B (strenge Isolierung als Einzelmaßnahme in einem der betreffenden Isoliersysteme) oder Gruppe C (Routinemäßige Hospitalpflege). Die Ergebnisse zeigen, daß die Inzidenz von Kontamination bzw. Kolonisierung durch neuerworbene Bakterienspezies bei strenger Isolierung allein und noch deutlicher bei einer strengen Isolierung und antibakterieller Dekontamination signifikant herabgesetzt werden konnten. Auch zeigte sich, daß in der Gruppe A weniger häufig schwere Infektionen auftraten. Noch höher signifikant schien das Auftreten von Lungeninfektionen in den beiden Gruppen A und B in Vergleich zu Gruppe C reduziert zu sein. Die Remissionsrate von Patienten mit akuter Leukämie war im Vergleich mit den Gruppen A und B zur Gruppe C höher (69%, 61%, 49%) obwohl dieses Ergebnis statistisch nicht signifikant war. Jedoch lassen die mikrobiologischen Untersuchungen und das Ergebnis der Studie vermuten, daß sowohl die Technik der protektiven Isolierung als auch die der antimikrobiellen Dekontamination verbessert werden müssen, um ein weiteres Absinken der Infektionsinzidenz bei gefährdeten Patienten zu erreichen.

Colonization resistance of the digestive tract and the spread of bacteria to the lymphatic organs in mice

Abstract After oral contamination of conventional mice with high doses of Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa the contaminant was recovered in abnormally high concentrations from the duodenum and caecum during the first few days. In this initial colonization phase, evidence of spread was obtained by culturing the cervical and mesenteric lymph nodes and spleen. Longer after contamination the intestinal concentration decreased to normal and spread stopped. In orally antibiotic-treated mice, the situation seen during the initial colonization phase in conventional mice occurred after a much lower oral contamination dose and persisted during the entire observation period of 2 weeks.

Determination of the colonization resistance of the digestive tract of individual mice

It has been shown that it is possible to investigate the colonization resistance in individual mice by determining the concentration of a certain contaminant (S.R.-E. coli) in the faeces during the first 4 days after contamination. Experimental contamination is contra-indicated in many cases such as in individuals with decreased resistance to infection. Particularly in this group, the value of the colonization resistance should be determined. It appeared to be possible to determine the colonization resistance in such individuals by quantitative biotyping of the Enterobacteriaceae species in the faeces on several consecutive days.

Bacteriological aspects of selective decontamination of the digestive tract as a method of infection prevention in granulocytopenic patients.

We describe the bacteriological results of a controlled clinical trial of selective decontamination of the digestive tract as a method of infection prevention in granulocytopenic patients. Selective elimination of Enterobacteriaceae and Pseudomonadaceae species was accomplished by the oral administration of nalidixic acid, co-trimoxazole, or polymyxin. Yeasts were eliminated selectively by amphotericin B or nystatin treatment. The drugs used in this study were chosen because of their capacities to selectively eliminate gram-negative rods and yeast without affecting the anaerobic part of the gut flora which is responsible for colonization resistance. Compared with the control group, the selectively decontaminated patients had significantly fewer (P less than 0.0005) gram-negative rods or yeasts or both in their throat swab cultures and in their feces. This reduction may explain the clinical effectiveness of selective decontamination.

Prevention of Infection in Acute Leukemia: A Prospective Randomized Study on the Efficacy of Two Different Drug Regimens for Antimicrobial Prophylaxis

SummaryIn a prospective study patients with acute leukemia undergoing remission induction therapy were randomized to receive either a regimen of non-absorbable antimicrobial drugs (colistin and neomycin) or of absorbable and non-absorbable drugs (trimethoprim-sulfamethoxazole [TMP-SMZ] and colistin) for antibacterial prophylaxis. For antifungal prophylaxis patients in both groups were given oral amphotericin B. The proportion of patients without acquired infections and the median of study time to the first acquired infection did not differ significantly between the two treatment groups (p>0.05). Septicemias occurred in nine out of 49 recipients of colistin and neomycin and in one out of 56 patients receiving TMP-SMZ and colistin (p=0.03). Localized infections and fever episodes without proven infections were equally distributed between the two groups. The incidence of febrile days and of days on parenteral antibiotic therapy was significantly lower in the group given TMP-SMZ and colistin (p<0.05). The duration of severe granulocytopenia and thrombocytopenia did not differ significantly between the two groups (p>0.05).ZusammenfassungIn einer randomisierten Studie wurde die Wirksamkeit von zwei Verfahren der Infektionsprophylaxe an Patienten mit akuten Leukämien untersucht. Die Patienten erhielten entweder eine Kombination nicht resorbierbarer Antibiotika (Colistin und Neomycin) oder eine Kombination resorbierbarer und nicht resorbierbarer antimikrobieller Substanzen (Trimethoprim-Sulfamethoxazol [TMP-SMZ] und Colistin). Zur Pilzprophylaxe wurde in beiden Gruppen Amphotericin B oral eingesetzt. Beide Gruppen unterschieden sich nicht signifikant hinsichtlich des Anteils von Patienten mit erworbenen Infektionen und hinsichtlich des Zeitintervalls bis zur ersten erworbenen Infektion (p>0,05). Septikämien traten bei neun von 49 Patienten auf, die Colistin und Neomycin erhielten, aber nur bei einem von 56 Patienten, die mit TMP-SMZ und Colistin behandelt wurden (p=0,03). Lokale Infektionen und Fieberepisoden ohne nachgewiesene Infektionen waren gleich häufig in beiden Gruppen. Die Häufigkeit von Fiebertagen und Tagen, an denen parenterale Antibiotika verabeicht wurden, war in der Gruppe, die TMP-SMZ und Colistin erhielt, signifikant geringer (p<0,05). Kein signifikanter Unterschied fand sich bei der Dauer der schweren Granulozytopenie und Thrombozytopenie (p>0,05).

Effect of twelve antimicrobial drugs on the colonization resistance of the digestive tract of mice and on endogenous potentially pathogenic bacteria

Twelve antimicrobial drugs were studied for their effect on the endogenous aerobic potentially pathogenic bacteria (Enterobacteriaceae, Streptococcus faecalis) in the intestines and on the colonization resistance (CR) of the digestive tract. Three subclasses of antimicrobial drugs could be recognized: (1) those which suppress the CR following low oral doses (rifamycin, penicillin V, cloxacillin, fenethicillin); (2) those in which the CR is suppressed only following relatively high oral doses (amoxycillin); and (3) those in which no obvious suppression of the CR was noticed even following substantial oral doses (nalidixic acid, cinoxacin, co-trimoxazole, oral cephalosporins, piv-mecillinam and doxycyclin). Some of the drugs in the third category were found to suppress endogenous Enterobacteriaceae (nalidixic acid, co-trimoxazole, piv-mecillinam and doxycyclin) and S. faecalis (doxycyclin) at dose levels at which they did not decrease CR.

THE DOSE AT WHICH NEOMYCIN AND POLYMYXIN-B CAN BE APPLIED FOR SELECTIVE DECONTAMINATION OF THE DIGESTIVE-TRACT IN MICE

Oral treatment of mice with various doses of neomycin or polymyxin B was performed in order to determine which dose caused substantial suppression of aerobic gram-negative rods. In addition the effect of the various doses on Streptococcus faecalis and on other factors of the colonization resistance (CR) of the digestive tract were studied. It was found that polymyxin B was effective in suppressing sensitive gram-negative bacteria following daily doses of 3.2 mg/mouse, and that even extremely high daily doses of 9.7 mg/mouse did not affect the CR. Neomycin was effective in suppressing Enterobacteriaceae species following oral daily doses of 5.4 mg/mouse. With this dose, however, the CR was somewhat decreased which was also evidenced by the increased concentration of beta-aspartylglycine in the faeces and the increased size (weight) of the caecum in these animals. Suppression of Str. faecalis was seen from doses of 24 mg/mouse on.

The colonization resistance of the digestive tract in different animal species and in man; a comparative study

The present study has attempted to determine the colonization resistance (CR) of the digestive tract by biotyping Enterobacteriaceae in four faecal samples per subject of five different animal species as well as man. The results indicate that the degree of bacterial contamination with Enterobacteriaceae from the environment may strongly influence the outcome. Both conventionally living chicken and man, showed a much wider range of the ‘confidence limits of the mean’ of the mean number of biotypes per faecal sample between individual subjects, than was found between subjects maintained under laboratory circumstances. Yet there appeared a statistically significant difference in CR between some of the animal species as a group. Man did not differ from monkeys, however, both differed from the rodents species studied. Monkeys differed also from dogs and the latter from rodents. It is concluded that the CR measured by determining the mean number of biotypes of Enterobacteriaceae can only be used for accurate comparison of the CR between subjects, if the ‘bacteriological environment’ is known; i.e. the sources of contamination.